Has anyone successfully accomplished this? I'm finding a few protocols in the lit for depleting mature B cells, but none seem to be validated for the pro B cells in RagKO mice on a B6 background. I'm feeling a bit too rushed to test different clones/dosing and I would appreciate any experience.
You should not have mature B cells from RagKO (RAG-1/2/) mice. Are you're mice Rag-1/2 KOs or just Rag-1 KOs? If the former, you probably need to screen the genotype of your mice and if the latter, maybe CD20 mAb like C2B8.
I have worked with RAG-1/2 KOs before but never screened for pro-B cells neither tried to deplete them so the latter suggestion is purely theoretical.
They're Rag1KO mice. I'm not sure what you mean there. Do Rag1/2 double KO's have a different phenotype with regards to the pro B cells?
I haven't looked at CD20 in the Rag's b/c I was under the impression that that marker comes up in pre B's and the Rag's arrest before that stage..
For sure there are no mature B cells but there are pro-B cells, both in single RAG ko and in dko. The answer depends on what you really want to do. If you want to specifically deplete the pro-B cell subpopulation without affecting any other BM subpopulations, I'm not sure you can make it easily with antibodies or other compounds. Hydroxyurea depletes cycling cells like proB cells but you would also deplete other hematopoietic cells (those in cycle). Alternatively, you can use different mouse models. If you do not want pro-B cells (and more mature B cells) in your mice, you can use IL7R ko mice. If your goal is really to induce the depletion of the pro-B cells, you can use the mb1-Cre mice (expression of Cre in all your B cell subpopulations), backcross them to Rosa-DTR, and then induce depletion of all the B cells (including the pro-B cells) with diphteria toxin. If you really need a RAGko background, then, backcross the mb1-Cre and Rosa-DTR with RAG ko. Tricky but feasible.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line